Generation of bioluminescent enzyme immunoassay for ferritin by single-chain variable fragment and its NanoLuc luciferase fusion

Abstract

Ferritin, widely present in liver and spleen tissue, is considered as a serological biomarker for liver diseases and cancers. The detection of ferritin may be an important tool in health diagnosis. In this study, 14 non-immunized chicken spleens were utilized to construct a single-chain fragment (scFv) phage library. After 4 rounds of panning, 7 unique clones were obtained. The optimal clone was further screened and combined with NanoLuc luciferase (Nluc) as a dual functional immunoprobe to bioluminescent enzyme immunoassay (BLEIA), which was twice as sensitive as its parental scFv-based double-sandwich enzyme-linked immunoassay (ds-ELISA). The cross-reactivity analysis revealed that the proposed methods were highly selective and suitable for clinical detection. To further verify the performance of the immunoassays, serum samples were tested by the proposed methods and a commercial ELISA kit, and there was a good correlation between the results. These results suggested that scFv fused with Nluc might be a powerful dual functional tool for rapid, practically reliable, and highly sensitive ferritin detection.

Keywords: Bioluminescent enzyme immunoassay; Biomarker; Ferritin; NanoLuc luciferase; Single-chain variable fragment.

Figure 1.

Seven unique amino acid sequences alignment of the scFvs against Ferritin. Framework regions (FR) and complementarity determining regions (CDR) of the light chain and heavy chain are indicated below the germline sequence.

Figure 2.

Calibration plots with ELISA (A) and BLEIA (B) based on scFv Fe38 and its Nluc fusion protein, respectively. Insert were the linear relationship between the absorbance/ luminous intensity and the concentration of ferritin. Error bars indicate standard deviations (n = 3).

Figure 3.

Specificity of ds-ELISA and BLEIA for ferritin. The microplate was coated with 100 μL polyclonal antibody (4 μg/mL) in CBS for overnight at 4 °C. Then 100 μL analytes (100 ng/mL ferritin or 1000 ng/mL others protein) were added for 1h at 37°C. The binding was detected with Fe38 scFv (HRP labeled mouse anti-HA antibody (1/10000) as tertiary antibody) or Fe38 scFv-Nlu. Error bars indicate standard deviations (n = 3).

Scheme 1.

The preparation of anti-ferritin scFv and BLEIA for detection of ferritin